Method of preferential labelling of a phycobiliprotein with an aminereactive dye for use in a multiple color assay and product for such use

ABSTRACT

A method for preparing phycobiliprotein/amine-reactive dye conjugates is disclosed in which the conjugates so prepared overcome the energy transfer/fluorescent quenching dilemma encountered in the use of prior art conjugates. A phycobiliprotein, for example, phycoerythrin or allophycocyanin, is conjugated with an amine-reactive dye, for example, Texas Red or carboxyfluorescein succinimidyl ester, in the presence of a selective salt which causes a hydrophobic intramolecular rearrangement of the phycobiliprotein thereby exposing more hydrophobic sites for binding to the amine-reactive dye. The conjugates prepared according to the invention are useful in multiple color fluorescence assays without requiring the use of multiple exciting sources.

RELATED APPLICATION

This is a continuation of application Ser. No. 08/227,573, filed on Apr.14, 1994 entitled "Method of Preferential Labelling of aPhycobiliprotein with an Amine-Reactive Dye for Use in a Multiple ColorAssay and Product for such Use", now abandoned, which is a continuationof Ser. No. 07/990,861, filed on Dec. 11, 1992 entitled "Method ofPreferential Labelling of a Phycobiliprotein with an Amine-Reactive Dyefor Use in a Multiple Color Assay and Product for such Use", nowabandoned, which is a Continuation-In-Part of application Ser. No.07/940,026, filed Sep. 3, 1992 entitled METHOD OF PREFERENTIAL LABELLINGOF A PHYCOBILIPROTEIN WITH AN AMINE-REACTIVE DYE FOR USE IN A MULTIPLECOLOR ASSAY AND PRODUCT FOR SUCH USE (now U.S. Pat. No. 5,272,257,issued Dec. 21, 1993); which is a Continuation-In-Part of Ser. No.07/526,387, filed May 21, 1990, entitled METHOD OF PREFERENTIALLABELLING OF A PHYCOBILIPROTEIN WITH A SECOND DYE FOR USE IN A MULTIPLECOLOR ASSAY AND PRODUCT FOR SUCH USE, now U.S. Pat. No. 5,171,846,issued Dec. 15, 1992. These applications and patents are owned by acommon assignee. Coulter Corporation, Hialeah, Fla.

FIELD OF THE INVENTION

This invention relates generally to electron donor-acceptor conjugatessuitable for use in multiple color assay methods, particularly to amethod of producing phycobiliprotein-dye conjugates suitable for suchuse.

BACKGROUND OF THE INVENTION

The technique of fluorescence was first introduced by Coons in 1941. Heused a blue fluorescing anthracene compound coupled to pneumococcusantiserum to detect bacterial antigens in tissue section. Subsequent tothis initial discovery, many fluorescing materials have beeninvestigated, but only two, the fluorochromes fluorescein and rhodamine,are widely used, particularly in the form of fluorescein isothiocyanate(FITC) and tetramethylrhodamine isothiocyanate (TRITC) respectively.FITC covalently binds to proteins at alkaline pH through the epsilon (ε)amino residues of lysine and through terminal amino groups. FITC'sadsorption maximum is at 490-495 nm and it emits its characteristicgreen color at 517 nm. TRITC likewise binds to proteins, has itsabsorption maximum at 541 nm and emits its characteristic red color at572 nm.

Fluorescence is the emission of light of one wavelength (color) by asubstance that is being irradiated by light of a different wavelength.The emitted light is always of lower energy, hence longer wavelength,then the incident light. In clinical use, the strength of thefluorescence is dependent on the efficiency with which the fluorochrometransforms incident light into emitted light, the amount of dye presentin the specimen under observation and the intensity of the incidentlight. The dye known as Texas Red (sulforhodamine 101 sulfonyl chlorideor sulforhodamine acid chloride) has previously been investigated forclinical use in conjugation with phycoerythrins, but major problems wereencountered. These problems were low fluorescent efficiency, inadequateenergy transfer from the phycoerythrin to Texas Red and the instabilityof the phycoerythrin-Texas Red conjugate. Phycoerythrin-Texas Redconjugates are desirable, however, because the overlap of theirabsorption and emission spectra have the potential to give a strongfluorescence signal.

Low fluorescent efficiency occurs whenever fluorescent chromophores arespatially adjacent to each other. It is usually called concentrationquenching. See R. P. Hughland, "Excited States of Biopolymers", R. F.Steins, Ed., p 47 (Plenum Press, New York, 1983). However, high levelsof labelling, resulting in chromophores being spatially adjacent to eachother, are required in order to assure adequate energy (electron)transfer from the phycoerythrin to the acceptor dye chromophore. The netresult is that the trade off required by the opposing effects results inless than optimal performance. Recently, A. N. Glazer et al. havecovalently linked a phycoerythrin to an allophycocyanin to produce ahighly fluorescent tandem conjugate with an energy transfer efficiencyof 90%. See A. N. Glazer et al., T.I.B.S, 9:423 (1984); Biophysics J.,43,386-386 (1983); and U.S. Pat. No. 4,542,104 (See also U.S. Pat. No.4,520,110 to L. Stryer et al. describing the use of phycobiliproteins asfluorescent probes for the analysis and separation of molecules andcells). However, forming a conjugate from two naturally occurringpigments derived from algae is much different from conjugating asynthetic dye such as Texas Red. In fact, the procedures usuallyfollowed for conjugating reactive dyes to proteins does not work withphycoerythrin-Texas Red. Using such procedures, one obtains a complexwith a low energy transfer efficiency at low levels of labelling orfluorescence quenching at high levels of labelling. Texas Red forms aconjugate with a phycoerythrin by reaction of its sulfonyl or acidchloride moiety with an amine group of phycoerythrin or otherphycobiliprotein.

Phycobiliprotein/amine-reactive dye conjugates are known and some, forexample, phycoerythrin-Texas Red conjugates, are commercially available.For example, the phycoerythrin-Texas Red conjugate known as DuoCHROME™is available bound to streptavidin from Becton Dickinson ImmunologySystems, Mountain View, Calif. (Catalog No. 9026). The availableconjugates, however, suffer from the fact that they do not have auniform phycoerythrin-Texas Red ratio throughout the individualconjugate members. There are present overlabelled and underlabelledspecies as well as species having the desired or optimum degree or rangeof labelling. Consequently, energy transfer/quenching problems can arisedepending upon the distribution of labelled species within the entiresample.

This invention solves the energy transfer/quenching problem encounteredin the preparation of phycobiliprotein/amine-reactive conjugates ingeneral by preferentially labelling sites close to the chromophoreregions of a phycobiliprotein with an amine-reactive dye and separatingoverlabelled and underlabelled conjugates from conjugates having thedesired degree of labelling by chromatographic methods; for example, byexploiting the differences in hydrophobic character of conjugates havingdifferent degrees of labelling.

SUMMARY OF THE INVENTION

A method is provided for preparing a phycobiliprotein/amine-reactive dye(PARD) conjugate which over comes the problems relating to the energytransfer/fluorescent quenching phenomenon encountered in suchconjugates. An amine-reactive dye, such as Texas Red or acarboxyfluoroscein succinimidyl ester, is reacted with aphycobiliprotein, such as a phycoerythrin or an allophycocyanin, in thepresence of a salt especially selected to cause an intramolecularrearrangement of the phycobiliprotein structure whereby to expose amultiplicity of sites in its hydrophobic region with which said dye canbind to form the desired conjugate. The reaction is controlled as to theanion of the selected salt, permitted time of reaction and temperature.Conjugates having the preferred degree ofphycobiliprotein/amine-reactive dye conjugation are separated fromoverlabelled and under-labelled conjugates by hydrophobic interactionchromatography. Alternatively, hydrophobic interaction chromatographymay be used to separate conjugates having the desired degree oflabelling from a reaction mixture which did not use the selective saltstaught herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the spectral differences between the PETR conjugateof the invention (A) and commercially available conjugates (B,C).

FIG. 2 illustrates the emission spectrum of theR-phycoerythrin/Cy5.18-OSu conjugate after excitation at λ=488 nm.

FIG. 3 illustrates the adsorption spectrum of theR-phycoerythrin/Cy5.18-OSu conjugate.

FIG. 4 illustrates the fluorescence spectrum for underconjugatedfraction of the PE-Cy5.18 conjugate, separated by hydrophobicinteraction chromatography.

FIG. 5 illustrates the fluorescence spectrum for overconjugated fractionof the PE-Cy5.18 conjugate, separated by hydrophobic interactionchromatography.

DETAILED DESCRIPTION OF THE INVENTION

The first feature of this invention, preferential site labelling, makesit possible to obtain a satisfactory level of energy transfer from aphycobiliprotein to an amine-reactive dye even at low levels of dyeconjugation by bringing the dye and the chromophore of thephycobiliprotein into close proximity. This is accomplished by makinguse of the hydrophobic tetrapyrrole (bilin) chromophores thatbiliproteins are known to possess. See R. McColl and D. Guard-Frier,Phycobiliproteins, Chapter 1, C.R.C. Press (1987). Specifically, whencertain anions commonly used in some "salting-out" processes are addedto a phycobiliprotein containing buffer solution, they cause thephycobiliprotein to undergo an intramolecular structural rearrangementwhich "opens-up" or "exposes" hydrophobic sites on the protein byreducing steric hindrance about the site. As a result of thishydrophobic intramolecular rearrangement, the sites close tochromophores can more readily react with a reactive dye, such as TexasRed, to form a conjugate. The common ions used in this process may beany of the common ions used in "salting-out" processes, such asphosphate, acetate, citrate, sulfate, tartrate and the like. Thepreferred anions are sulfate, phosphate and acetate. The most preferredanion is sulfate because it has little or no effect on the pH of thesolution. The exact amount of anion required in a given reaction isdependent on the particular phycobiliprotein undergoing reaction. Forexample, when using the sulfate in a phycoerythrin-Texas Red (PETR)conjugation reaction, it was found that an anion concentration in therange of about 1% to about 4% in the reaction solution resulted in aPETR conjugate having significantly improved energy transfer efficiencyas compared to a PETR control conjugate prepared in the absence of apreferred anion. On the other hand, allophycocyanin requires the use ofabout 8% to 12% sodium sulfate. Using the principles taught herein, theoptimal concentration of the selected salt can easily be determined.Overall, the optimal concentrations will range between 1% and about 20%.

The phycobiliprotein and the amine-reactive dye are reacted together ata pH greater than 7 for a time in the range of 10 minutes and at atemperature of about 4° C. to about 25° C. prior to sampling todetermine if an overall adequate phycobiliprotein-dye conjugation ratiohas been reached. The preferred pH is greater than 8 and less than 12.The determination is carried out by chromatographically removing excessdye from a sample of the reaction mixture and spectroscopicallydetermining an absorbance ratio, A_(x) /A_(y), defined as the ratio ofthe intensity of the maximum absorption of the phycobiliprotein dividedby the intensity of the maximum adsorption of the amine-reactive dye.For a PETR conjugate, A_(x) /A_(y) is A₅₆₅ /A₅₉₅. If the value of A₅₆₅/A₅₉₅ is in the range of 2.9 to 3.2, the reaction mixture is quenchedand excess dye is removed. The A_(x) /A_(y) value will differ fordifferent PARD conjugates. The removal of the excess dye simultaneouslyremoves excess salts such as the sodium sulfate preferably used toexpose a phycobiliprotein's hidden hydrophobic sites.

An excess of amine-reactive dye is used in the claimed method. Theinitial molar ratio of amine-reactive dye to phycobiliprotein in thereaction mixture is in the range of about 5:1 to about 30:1.

A phycobiliprotein/amine-reactive dye conjugate is formed by reaction ofan amino group on the phycobiliprotein with a reactive group present onthe amine-reactive dye. For example, a phycoerythrin-Texas Red conjugateis formed by reaction of an amino group on phycoerythrin with thesulfonyl or acid moiety of Texas Red. The reactivity of phycobiliproteinamino groups is well known. For example, small biomolecules such asbiotin have been attached to phycobiliproteins by reaction with anappropriate activated ester or sulfonyl chloride derivative. Thereaction between phycobiliproteins and amine-reactive dyes as taughtherein, for example, the reaction between phycoerythrin and Texas Red,is analogous to the biotin reaction and to the reactions of fluoresceinisothiocyanate with the ε-amino residues of lysine and terminal aminogroups previously mentioned. While any moiety reactive with amines maybe used according to the invention, the preferred reactive moietiespresent on the amine-reactive dye are selected from the group consistingof sulfonic and carboxylic acids and their acid chlorides and esters.Specific examples of such dyes include 5- or 6-carboxyl-x-rhodamainesuccinimidyl esters, sulforhodamine 101 sulfonyl chloride, Lisseminerhodamine B sulfonyl chloride. Compounds such asfluorescein-5-isothiocyanate and fluorescein-6-isothiocyanate, arefurther examples of amine-reactive dyes.

Phycobiliproteins is general may be used according to the invention. Inaddition to phycoerythrin and allophycocyanin used in the Examplesherein, C-phycocyanin, R-phycocyanin and phycoerythrocyanin, amongothers, may be reacted as described herein.

The separation of over-labelled and under-labelled PARD conjugatespecies from those having the desired degree of labelling wasaccomplished using hydrophobic interaction chromatography with anappropriate column medium like butyl TOYOPEARL™ (Toso Haas,Philadelphia, Pa.). The PARD conjugate produced by this method can beused in conjunction with an antibody to stain different types of cell.The cells so stained will be dependent upon the choice of antibody. Theimportance of the invention lies in the fact that PARD conjugatesprovide for an additional color in fluorescence analysis with the use ofonly a single excitation wavelength, which wavelength is determined bythe choice of the amine-reactive dye. For example, FITC, R-phycoerythrinPETR and APC-FSE can be excited with a single excitation wavelength(laser) of 488 nm to emit maximally at about 525, 575, 612 and 660 nmrespectively. As a result of this feature, the expense of multipleexcitation sources is eliminated.

PREFERRED EMBODIMENTS OF THE INVENTION Example 1 Reaction ofPhycoerythrin and Texas Red

In a typical reaction, a purified R-phycoerythrin (PE) solution 3.0 gPE, 45.04 ml solution; PE concentration is 66.6 mg/ml in 2 mM EDTA-PBS(PBS=Phosphate Buffered Saline)! was cooled in ice-bath and treateddropwise, with stirring, with an ice-cold solution of PBS containing 2mM EDTA (29.25 ml), 20% Na₂ SO₄ (pH 7.0, 6.0 ml) and 1M potassium borate(pH 9.80, 30 ml). To the resulting mixture was added with vigorousstirring and at 4° C. a 25-fold molar excess of Texas Red (20 mg/ml inanhydrous dimethylformamide). The reaction was monitored by drawing 10μL samples periodically and removing excess dye on a 0.5-2 ml Sephedex®G-50 column in PBS. The protein containing peak was collected and itsA₅₆₅ /A₅₉₅ value determined spectrophotomet If the A₅₆₅ /A₅₉₅ valuesremain above 3.2, even after 30 minutes or more of reaction time, afurther aliquot of Texas Red solution of 1-5 times the initial PEconcentration was added to the reaction mixture.

When A₅₆₅ /A₅₉₅ value fell below 3.2, preferably in the range of2.9-3.2, the reaction may be quenched by addition of an one-hundred foldmolar excess of a quenching agent glycine to the reaction mixture.Typical quenching agents are glycine, hydroxylamine hydrochloride,ethanolamine and lysine among others. Excess reactive dye was nextremoved by passing the reaction mixture through a SEPHADEX® G-50 columnin PBS, 2 mM EDTA. The phycoerythrin-Texas Red conjugate, in the proteinpeak, was then chromatographically factionated on a butyl 650Mchromatographic column by eluting with a reverse gradient (3% to 0%) ofsodium sulfate in 100 mM potassium phosphate solution containing 2 mMEDTA at pH 7.0±0.1. Butyl 650M is an abbreviation of butyl TOYOPEARL™650M available from Toso Haas). Chromatographic fractions having thedesired emission characteristics (high energy transfer and high quantumefficiency) were pooled, concentrated, dialyzed against PBS, 2 mM EDTAand reconcentrated to give a purified phycoerythrin-Texas Red conjugate.The purified PETR conjugate was used as a marker in fluorescentimmunoassays. The PETR marker can be conjugated to protein-likesubstances such as antibodies and streptavidin using methods known inthe art. FIG. 1 illustrates the emission spectra differences between thePETR conjugate made according to the invention and commerciallyavailable conjugates (B, Southern Biotechnology Associates; C,Becton-Dickinson).

Example 2 Reaction of Allophycocyanin (APC) with a CarboxyfluoresceinSuccinimidyl Ester (FSE)

6.25 ml (292.4 mg) APC solution (46.748 mg/ml in PBS, 2 mM EDTA) wastreated by dropwise addition, with stirring, of a solution resultingfrom mixing PBS, 2 mM EDTA (6.17 ml), 1M potassium borate of pH 9.80(7.3 ml) and 20 wt % sodium sulfate of pH 7.0 (8.76 ml). A 14-foldexcess of FSE (25 mg/ml in anhydrous dimethylforamide) was added to theAPC solution at room temperature (about 22° C.). After 30 minutes, a 50μL sample of the reaction mixture was withdrawn, excess dye removed on aSEPHADEX® G-50 column in PBS which was 2 mM in EDTA, and the proteinA₄₉₆ /A₆₅₂ value for the protein peak was checked.

If the A₄₉₆ /A₆₅₂ ratio is less than 0.5 after 60 minutes or morereaction time, an additional aliquot of FSE was added to the reactionmixture. When an A₄₉₆ /A₆₅₂ value in the range of 0.5-0.7 is achieved,the reaction is stopped by removing excess dye on a SEPHADEX® G-50column in PBS, 2 mM EDTA. Alternatively, a quenching agent is added tothe reaction mixture prior to passage through the Sephadex® column. Theprotein peak is collected and the APC-FSE conjugate mixture is furtherfractionated using hydrophobic interaction chromatography on a butyl650S (Toso Haas) column using a reverse gradient (4% to 0%) sodiumsulfate in 100 mM potassium phosphate, 2 mM EDTA of pH 7.0±0.1, followedby steps of 50 mM potassium phosphate, 2 mM EDTA of pH 7.0±0.1, andlastly, PBS, 2 mM EDTA. Fractions showing high energy transferefficiency and high fluorescence were found to have an A₄₉₆ /A₆₅₂ ratioof about 0.4-0.6. These were pooled, dialyzed against PBS, 2 mM EDTA andconcentrated. The yield was 54%. The conjugate was used without furtherpurification.

Example 3 APC-FSE Conjugate Prepared Without Using Selected Salts

APC and FSE are reacted as in Example 2, but without the addition of theselected salt. The reaction time is extended within the range of 0.5 to5.0 hours after the addition of FSE to APC is completed. The reactionmixture is then passed through a SEPHADEX® G-50 column and issubsequently fractionated by hydrophobic interaction chromatography.Fractions having A₄₉₆ /A₆₅₂ in the range of 0.4-0.6 are collected,dialyzed and concentrated, and may be used without further purification.The yield is lower than in Example 2 where selected salts were used andis estimated to be about 35% at a maximum.

Example 4 Reaction of R-Phycoerythrin with the Amine-Reactive DyeCy5.18-OSu

1 ml (50 mg) of purified R-phycoerythrin solution (PE, 50 mg/ml 2 mMEDTA), cooled to 0° C. in an ice bath, was treated by dropwise addition,with stirring, with a 0° solution resulting from mixing a 1M borate-PBSsolution (pH 9.8, 3.125 ml), 2 mM EDTA (4.45 ml) and 20% Na₂ SO₄ (pH7.0, 0.625 ml). A 10-fold molar excess of Cy5.18-OSu (hereafter CY, 1mg/ml in PBS, 2 mM EDTA, available as Cy5™ Cy5.18-OSu(5,5'-Bis-sulfo-1,1'-bis(ε-carboxypentynyl)-3,3,3',3'-tetramethylindodicarbocyaninedisuccinimidyl ester) from Biological Detection Systems, Inc.,Rockville, Md.;) was added to the resulting R-phycoerythrin solution.Reaction progress was monitored by periodically taking a 50 μL sample ofthe reaction mixture, desalting the sample on a Sephadex® G-50 column inPBS which is 2 mM in EDTA, collect- ing the protein peak andspectroscopically determining the protein A₅₆₅ /A₆₆₅. If the value ismore than 2.3 after 10 minutes of reaction, another 1- to 5-fold excessof CY, relative to the initial PE quantity, was added to the reactionmixture. About 1-4% of a selected salt is used in the preparation ofPE/CY.

When A₅₆₅ /A₆₅₁ value is in the range of 1.6-2.3, the reaction mixturewas desalted on a SEPHADEX® G-50 column in PBS which is 2 mM in EDTA.Alternatively a quenching agent such as hydroylamine or 2-aminoethanolis added to the reaction mixture before desalting on SEPHADEX® G-50 asdescribed. The protein peak is collected and the PE-CY conjugate isfurther fractionated using hydrophobic interaction chromatography on aButyl 650S column using a reverse gradient (4.5% to 0% w/v) sodiumsulfate solution which is 100 mM in potassium phosphate, 2 mM EDTA andpH 7.0±0.1. The chromotographic fractions were checked for emissions at575 nm (PE) and 665 nm (CY) using a 488 nm excitation source. Thefractions having high energy transfer efficiency (E₅₇₅ /Em₆₆₅ ≦0.15) andhigh energy transfer efficiency were combined, concentrated, dialyzedagainst PBS, 2 mM EDTA and reconcentrated.

The spectrum of the product from a typical preparation, FIG. 2, showsabout 93-96% energy transfer efficiency (Em₅₇₅ /Em₆₆₅ =about 0.04 toabout 0.07). The PE/CY product can then be conjugated to proteins suchas antibodies by method known to those skilled in the art and used invarious analytical methods such as flow cytometry.

                  TABLE 1                                                         ______________________________________                                        Peaks & Valleys in Absorption                                                 Spectrum of PE/CY (FIG. 3).                                                   PEAK                    VALLEY                                                λ ABS            λ                                                                             ABS                                            ______________________________________                                        651.0    0.398          779.0  -0.002                                         607.0    0.175          618.0  0.166                                          568.0    0.928          592.0  0.151                                          497.0    0.647          510.0  0.483                                          369.0    0.104          425.0  0.025                                          278.0    0.184          324.0  0.059                                                                  263.0  0.160                                          ______________________________________                                    

I claim:
 1. A method of producing a phycobiliprotein-amine reactive dyeconjugate which method comprises reacting the phycobiliprotein with theamine reactive dye, desalting the reaction mixture to remove the dye notconjugated with the phycobiliprotein, and further separating theconjugate by hydrophobic interaction chromatography in order to separateout phycobiliprotein underlabelled and overlabelled with amine reactivedye from that having the desired degree of labelling with the aminereactive dye.
 2. The method according to claim 1 wherein thephycobiliprotein is phycoerythrin and the amine reactive dye is TexasRed.
 3. The method according to claim 1 wherein the phycobiliprotein isallophycocyanin and the amine reactive dye is carboxyfluoresceinsuccinimidyl ester.
 4. The method according to claim 1 wherein thephycobiliprotein is phycoerythrin and the amine reactive dye is5,5'-Bis-sulfo-1,1'-bis(ε-carboxypentynyl)-3,3,3',3'-tetramethylindodicarbocyaninedisuccinimidyl ester.
 5. A method of producing a phycoerythrin-aminereactive dye conjugate which method comprises reacting the phycoerythrinwith the amine reactive dye in the presence of 1 to 20% salt, desaltingthe reaction mixture in order to remove the dye not conjugated with thephycoerythrin and further separating the conjugate by hydrophobicinteraction chromatography in order to separate out phycoerythrinunderlabelled and overlabelled with amine reactive dye from that havingthe desired degree of labelling with the amine reactive dye.
 6. Themethod of claim 5 wherein the amine reactive dye is5,5'-Bis-sulfo-1,1'-bis(ε-carboxypentynyl)-3,3,3',3'-tetramethylindodicarbocyaninedisuccinimidyl ester.
 7. The method of claim 5 wherein said salt is onewhose anion is selected from the group consisting of phosphate, sulfate,acetate, citrate and tartrate ions.